Hypoxia Inducible Factor-1α and Microvessel Density as Angiogenic Factors in Bilharzial and Non-Bilharzial Bladder Cancer

Abstract

Hypoxia inducible factor (HIF)-1alpha is a critical regulatory protein of cellular response to hypoxia and is closely related to angiogenic processes. Microvessel density (MVD), a measure of tumor angiogenesis has been shown to be predictive of progression and poor prognosis in bladder urothelial carcinoma. Most research has relied on measuring HIF-1alpha by immunohistochemistry on tissue sections and studying its prognostic value. However, no study has investigated HIF-1alpha expression by ELISA technique in association with angiogenesis in bilharzial and nonbilharzial bladder carcinoma. The primary objective of this pilot case control study was to measure HIF-1alpha level by ELISA technique in voided urine samples in a trial to find a diagnostic applicability in patients with bilharzial and nonbilharzial bladder carcinoma. Secondary objectives were assessment of MVD in relation to HIF-1alpha positivity as well as correlating them with clinicopathological variables to get insight in their potential prognostic and predictive value in bladder cancer. Voided urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (Group 1: n = 39), urological patients without urothelial carcinoma (Group 2: n = 15), and healthy volunteers (Group 3: n = 15). All underwent serological assessment of bilharzial antibody, quantitative measurement of HIF-1alpha by ELISA in urothelial cells of voided urine samples and urine cytology. MVD was calculated by immunohistochemical staining of endothelial cells with CD34 on tumor tissue paraffin sections. There was a statistically significant difference between benign and malignant groups regarding HIF-1alpha positivity rate (p < 0.001). Urinary HIF-1alpha best cut-off was determined using receiver operating characteristic curves to discriminate between malignant and nonmalignant groups (21.7 ng/mg protein) at 82.1% sensitivity and 63.3% specificity. The sensitivity of urine cytology was increased on combination with HIF-1alpha from 53.8% to 92.8%. In the malignant group, MVD revealed a high score in 70% and a low score in 30% of cases compared to 0% and 100%, respectively, in the benign group. The difference was highly significant (p < 0.001). There was no significant relationship between HIF-1alpha positivity rate or MVD and stage, as well as histologic grade of the tumor (p > 0.05) denoting no prognostic significance. HIF-1alpha can be reliably and quantitatively measured in urine of bladder cancer patients, improving the sensitivity and specificity of urine cytology for the diagnosis of bladder cancer. Independent studies, however, will be required on larger cohorts to validate these findings.